微小RNA-4287调控人软骨细胞聚蛋白多糖酶-2表达的研究  

MicroRNA-4287 regulates the expression of aggrecanase-2 in human chondrocytes

作　　者：孙红 张志奇[2] 毛谷平 黄志宇[2] 余宝禧 张程云 傅明[2] Sun Hong;Zhang Zhiqi;Mao Guping;Huang Zhiyu;Yu Baoxi;Zhang Chengyun;Fu Ming(Department of Orthopedics,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;Department of Joint Surgery,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]贵州医科大学附属医院骨科,贵阳550004 [2]中山大学附属第一医院关节外科,广州510080

出　　处：《中华实验外科杂志》2019年第5期906-908,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81572171).

摘　　要：目的探讨微小RNA(miRNA,miR)-4287对人软骨细胞聚蛋白多糖酶-2(ADAMTS5)的调控作用及其机制。方法从膝关节软骨组织中分离得到软骨细胞,白细胞介素(IL)-1β诱导软骨炎症模型,过表达或抑制细胞中miR-4287水平,实时荧光定量聚合酶链反应(FQ-PCR)及蛋白质印迹法(Western blot)分析miR-4287和ADAMTS5 mRNA及蛋白表达,双荧光素酶报告基因方法验证miR-4287与ADAMTS5 mRNA 3’非翻译区(UTR)的结合效应。结果miR-4287在人骨关节炎(OA)软骨组织中表达较正常软骨组织减少(0.354±0.167比1.074±0.507;t=3.010,P<0.05),而ADAMTS5 mRNA表达升高(21.019±7.044比1.902±2.081;t=-5.820,P<0.01)。与未刺激组比较,IL-1β刺激软骨细胞12 h后,miR-4287表达降低(0.454±0.095比1.010±0.166;t=11.410,P<0.01),而ADAMTS5 mRNA表达增多(5.067±0.994比1.006±0.137;t=7.306,P<0.05)。转染miR-4287模拟物48 h后,软骨细胞内ADAMTS5 RNA(0.620±0.366比1.023±0.269;t=6.047,P<0.05)和蛋白表达均降低;而转染miR-4287抑制剂后,软骨细胞内ADAMTS5 RNA(2.502±0.625比1.018±0.226;t=5.637,P<0.05)和蛋白表达均升高。结合位点为野生型时,过表达miR-4287能降低报告载体的荧光素酶活性(0.789±0.088比1.018±0.226;t=2.806,P<0.05);结合位点突变后,过表达miR-4287能则不能改变报告载体的荧光素酶活性(1.000±0.006比1.014±0.065;t=0.364,P>0.05)。结论miR-4287是人软骨细胞ADAMTS5表达的直接调控因子,可能在软骨基质代谢稳态维持方面具有重要的作用。Objective This study was undertaken to determine whether aggrecanase-2(ADAMTS5)can be regulated by microRNA(miRNA,miR)-4287.Methods Chondrocytes were separated from knee cartilage and induced with interleukin-1 beta(IL-1β).The expression of miR-4287 and ADAMTS5 was quantified by real time fluorescence quantitive polymerase chain reaction(FQ-PCR)by SYBR Green assay and Western blotting.Luciferase reporter assay was used to validate the direct interaction between miR-4287 and putative site in the 3’untranslated region(3’UTR)of ADAMTS5 mRNA.Results Compared with normal cartilages,the expression of miR-4287 was significantly lower(0.354±0.16 vs.1.074±0.507;t=3.010,P<0.05)and the expression of ADAMTS5 mRNA was higher(21.019±7.044 vs.1.902±2.081;t=-5.820,P<0.01)in osteoarthritic knee articular cartilages.Simulated with IL-1βled to a reduction in miR-4287 expression(0.454±0.095 vs.1.010±0.166;t=11.410,P<0.01)and upregulation in ADAMTS5 expression(5.067±0.994 vs.1.006±0.137;t=7.306,P<0.05).ADAMTS5 expression was suppressed in mRNA(0.620±0.366 vs.1.023±0.269;t=6.047,P<0.05)and protein by transfection with miR-4287 mimics and induced in mRNA(2.502±0.62 vs.1.018±0.226;t=5.637,P<0.05)and protein by transfection with miR-4287 inhibitor on the contrary.MiR-4287 overexpression directly suppressed the luciferase activity of a reporter construct containing the 3’UTR of ADAMTS5 mRNA(0.789±0.088 vs.1.018±0.226;t=2.806,P<0.05).However,treatment with miR-4287 mimics failed to influence the luciferase activity of this reporter construct(1.000±0.006 vs.1.014±0.065;t=0.3643,P>0.05).Conclusion MiR-4287 is a direct regulator of ADAMTS5 expression in human chondrocytes that may play an important role in the regulation of human cartilage homeostasis.

关 键 词：微小RNA-4287 骨关节炎 白细胞介素-1Β 聚蛋白多糖酶-2 

分 类 号：R68[医药卫生—骨科学]